1. Field of the Invention
The invention relates generally to the removal of proteinase inhibitors from plant sources and, more specifically, to the removal of Kunitz and Bowman-Birk trypsin inhibitors and carboxypeptidase inhibitors from proteins extracted from potato tubers.
2. Background of the Prior Art
Proteins that inhibit proteolytic enzymes are often found in high concentrations in many seeds and other plant storage organs. Inhibitor proteins are also found in virtually all animal tissues and fluids. These proteins have been the object of considerable research for many years because of their ability to complex with and inhibit proteolytic enzymes from animals and microorganisms. The inhibitors have become valuable tools for the study of proteolysis in medicine and biology. Protease inhibitors are of particular interest due to their therapeutic potentials in controlling proteinases involved in a number of disorders such as pancreatitis, shock, and emphysema, and as agents for the regulation of mammalian fertilization. Potato tubers are a rich source of a complex group of proteins and polypeptides that potently inhibit several proteolytic enzymes usually found in animals and microorganisms. In particular, potato inhibitors are known to inhibit human digestive proteinases, and thus have application in the control of obesity and diabetes.
Proteinase inhibitors found in plants are typically polypeptides and proteins that are composed entirely of L-amino acids through peptide bonds. These proteinase inhibitors differ significantly in their properties. The association of natural proteinase inhibitors with the proteinases that they inhibit is strong at neutral pH, and association constants are usually in the range of 107-1014Mxe2x88x921. Such associations are pH-dependent, and they decrease rapidly as the pH is lowered from neutrality to 3 (Ryan, C. A., and Walker-Simmons, M. 1981. Plant Proteinase. In The Biochemistry of Plants, V6, pp. 321-350, Academic Press).
Plant proteinase inhibitors generally are quite stable molecules and are often resistant to heat, pH extremes, and proteolysis by proteinases, even by those they do not inhibit. This stability has been attributed in part to the high degree of cross-linking through disulfide bridges. Other, non-covalent interactions also contribute significantly to the stability of the inhibitors. For example, protease inhibitor I from potatoes is a powerful chemotrypsin inhibitor that, while stable in solution at 80xc2x0 C. for several minutes (Melville, J. C., and Ryan, C. A. Chemotrypsin inhibitor I from potatoes. J. Microb. Chem. 247: 3445-3453, 1972), contains only one disulfide bond per monomer unit (MW xcx9c8,300) that can be reduced and carboxymethylated without loss of inhibitory activity (Plunkett, G., and Ryan, C. A. Reduction and carboxamidomethylation of the single disulfide bond of proteinase inhibitor I from potato tubers. Effects on stability, immunological properties, and inhibitory activities. J. Biol. Chem., 255: 2752-2755, 1980).
Several proteinases exhibit substrate specificity, whereas others, such as papain, have broad substrate specificity. Specific proteinase inhibitor families were identified for each of the four mechanistic classes of proteolytic enzymes, i.e., serine, cysteinyl, aspartyl and metallo-proteases (Ryan, C. A. Proteinase Inhibitors. In The Biochemistry of Plants, V6, pp.351-370, Academic Press). In other circumstances, identical abundant proteins were capable of inhibiting enzymes of various families and have very different substrate specificity, such as the inhibitors of both proteinases and xcex1-amylases which were isolated from cereal seeds (Campos, F. A. P., and Richardson, M. The complete amino acid sequence of xcex1-amylases/trypsin inhibitor from seeds of ragi (Indian finger millet; Eleusine coracana Goertn.). FEBS Lett., 152: 300-304, 1983; Campos, F. A. P., and Richardson, M. The complete amino acid sequence of xcex1-amylases/trypsin inhibitor from seeds of ragi (Indian finger millet; Eleusine coracana Goertn.). FEBS Lett., 167: 221-225, 1984).
Two broad classes of protease inhibitor superfamilies have been identified from soybean and other legumes with each class having several isoinhibitors. Kunitz-type inhibitor is the major member of the first class whose members have 170-200 amino acids, molecular weights between 20,000 and 25,000, and act principally against trypsin. Kunitz-type proteinase inhibitors are mostly single chain polypeptides with 4 cysteines linked in two disulfide bridges, and with one reactive site located in a loop defined by disulfide bridge. The second class of inhibitors contains 60-85 amino acids, has a range in molecular weight of 6000-10,000, has high proportion of disulfide bonds, is relatively heat-stable, and inhibits both trypsin and chemotrypsin at independent binding sites. Bowman-Birk inhibitor is an example of this class.
Kunitz inhibitor is capable of inhibiting trypsin derived from a number of animal species as well as bovine chemotrypsin, human plasmin, and plasma kallikrein. The cationic form of human trypsin, which accounts for a majority of trypsin activity, is only weakly inhibited by the Kunitz inhibitor, whereas the anionic form is fully inhibited.
The Bowman-Birk inhibitor is a 71 amino acid chain protein with 7 disulfide bonds characterized by its low molecular weight of about 8000 (in non-associated monomers), high concentration (about 20%) of cystine, high solubility, resistance to heat denaturation and having the capacity to inhibit trypsin and chymotrypsin at independent inhibitory sites.
The major effects of proteinase inhibitors in animal diets include growth depression and pancreatic hypertrophy. Resistance of raw soybean protein to proteolysis, low levels of sulfur-containing amino acids in soybean proteins, and lower digestibility, absorption, and utilization of available nitrogen from the small intestine due to the presence of proteinase inhibitors, all appear to contribute to growth depression.
Proteinase inhibitors extracted from potatoes have been distinguished into two groups based on their heat stability. The group of inhibitors that is stable at 80xc2x0 C. for 10 minutes have been identified as inhibitor I (mol. wt. 39,000) (Melville et al.), carboxypeptidase inhibitor (CPI) (mol. wt. 4,100) (Ryan, C. L., Purification and properties of a carboxypeptidase inhibitor from potatoes. J. Biol. Chem. 249: 5495-5499, 1974), inhibitors IIa and IIb (mol. wt. 20,700) (Bryant, J., Green, T. R., Gurusaddaiah, T., Ryan, C. L. Proteinase inhibitor II from potatoes: Isolation and characterization of its protomer components. Biochemistry 15: 3418-3424, 1976), and inhibitor A5 (mol. wt. 26,000).
Separation of proteinase inhibitor I by ion exchange chromatography on sulfoethylcellulose in the presence of 0.1 M formic acid in 8 M urea resolved two major and two minor inhibitor protomers. Reassociation by dilution to the tetramer form resulted in two major protomers. The first protomer was shown to be a powerful inhibitor of both chymotrypsin and trypsin. The second protomer was shown to strongly inhibit chymotrypsin but only weakly inhibit trypsin. All four purified promoters resolved from Inhibitor I can be reassociated either individually or hybridized with each other to form tetrameric isoinhibitors. All of the tetrameric inhibitor I species prepared from each of the four protomeric types have glutamic acid at the NH2 terminal. However, they differ from each other in amino acid composition, electrophoretic mobility, reactivity with chymotrypsin and trypsin, and digestibility with pepsin.
Proteinase inhibitor II, an inhibitor of chemotrypsin and trypsin, which are serine proteases, is also a heat stable protein. It has a dimeric molecular weight of 21,000. Four monomeric isoinhibitor species of molecular weight 10,500 comprise inhibitor II and have been isolated by chromatography in the presence of urea. Upon removal of the urea, each monomeric species dimerized to yield homogenous dimers. The three major protomer species, called B, C, and D were found to have similar molecular weights and amino acid compositions, and each has an N-terminal alanine residue. Reconstituted dimers possess two binding sites for bovine xcex1-chymotrypsin, indicating that each monomer possesses one binding site for this enzyme. Significant differences have been noted among the reconstituted dimers in their isoelectric points, immunoelectrophoretic mobilities, ion-exchange properties, and their inhibitory reactivities against trypsin. The properties of the inhibitor II dimeric species are similar but not identical to inhibitors IIa and IIb reported from Japanese potatoes, indicating the existence of intervarietal, as well as intravarietal, differences among potato tuber inhibitor II isoinhibitors (Bryant et al.).
Protease inhibitor II is composed of two sequence repeats. It contains two reactive site domains. The role of the two reactive sites in the inhibition of trypsin and chemotrypsin has been evaluated. The first reactive site inhibits only chymotrypsin (Ki=2 nM), and this activity is very sensitive to mutations. The second reactive site strongly inhibits trypsin (Ki=0.4 nM) and chemotrypsin (Ki=0.9 nM), and is quite stable towards mutations (Beekwilder, J., Schipper, B., Bakker, P., Bosch, D., and Jongsma, M. Characterization of potato proteinase inhibitor II reactive site mutants. Eur. J. Biochem., 267: 1975-1984, 2000).
In addition to inhibitor I and inhibitor II, several low molecular weight inhibitors have been detected in potato. Among them are the carboxypeptidase inhibitor, which has been extensively characterized (Bryant et al., 1976 and Iwasaki, T., Kijohara, T., and Yoshikawa. J. Biol. Chem. (Tokyo) 72: 1029, 1972) and at least three inhibitors of serine proteinases. The amino acid sequences of two low molecular weight serine proteinase inhibitors from Russet Burbank potatoes have been determined. One of those, a chemotrypsin inhibitor, is a peptide of 52 amino acid residues, while the second inhibitor, which is specific for trypsin, contains 51 amino acid residues. These peptides are highly homologous, differing at only nine positions. At position 38, the chymotrypsin inhibitor possesses leucine and the trypsine inhibitor an arginine. The inhibitors are also homologous with potato inhibitor II and with an inhibitor previously isolated from eggplants (Hass, et al., 1982).
U.S. Pat. No. 5,187,154 describes a method for the diagnosis and the treatment of individuals with diabetes or at risk to develop diabetes mellitus. In particular, gastric emptying determinations are used to assess risk. Risk or early symptoms associated with subsequent development of diabetes mellitus may be controlled or alleviated by delaying gastric emptying, which was achieved by the administration of cholecystokinin.
U.S. Pat. No. 4,906,457 describes compositions and methods for reducing the risk of skin cancer. The described compositions included at least one effective protease inhibitor. Preferred protease inhibitors included serine protease inhibitors and metallo-protease inhibitors. The protease inhibitors were preferably included in concentrations ranging from approximately 10 picograms to 10 milligrams per milliliter of the skin-applicable topical mixtures. The topical mixtures preferably included a suitable topical vehicle such as a cream, lotion, or ointment. One class of anti-carcinogenic skin treatment compositions of this invention preferably included the desired protease inhibitors in combination with a suitable sunscreen agent or agents, such as para-amino benzoic acid, to provide particularly advantageous compositions for reducing the risk of sunlight-induced skin cancer.
When applied to mouse epidermal JB6 cells, proteinase inhibitors I and II from potatoes blocked the UV induced transcription factor activator protein-1 (AP-1), which has been shown to be responsible for the tumor promoter action of UV light in mammalian cells. The inhibition appears to be specific for UV induced signal transduction for AP-1 activation. Furthermore, the inhibition of UV induced AP-1 activity occurs through a pathway that is independent of extracellular signal-regulated kinases and c-jun N-terminal kinases as well as P38 kinases (Huang, C., Ma, W. Y., Ryan, C. A., Dong, Z. Proteinase inhibitors I and II from potatoes specifically block UV-induced activator protein-1 activation through a pathway that is independent of extracellular signal regulated kinases, c-jun N-terminal kinases, and P38 kinase. Proc. Natl. Acad. Sci., US, 94: 11957-11962, 1997).
U.S. Pat. No. 4,491,578 describes a method of eliciting satiety in mammals through the administration of an effective amount of a trypsin inhibitor. The method was based on the postulate that the enzyme trypsin, normally secreted by the pancreas, constitutes a negative feedback signal for cholecystokinin secretion that in turn comprises a putative satiety signal. Thus, the effect of the trypsin inhibitor is to increase the concentration of cholecystokinin secretion advancing the sensation of satiety resulting in a consequent decrease in food intake and, over time, body weight.
The effect of PI2 extracted from potatoes, which increases CCK release, or food intake was examined in 11 lean subjects. Five minutes before presenting them with a lunchtime test meal, volunteers received 1.5 g PI2 in a high protein soup vehicle (70 kcal), the soup vehicle alone, or a no-soup control, according to a double blind, within subject design. The consumption of the soup alone led to a non-significant 3% reduction in energy intake. The addition of 1.5 g PI2 to the soup significantly reduced energy intake by additional 17.5%. Pre-meal ratings of motivation to eat and food preferences did not predict the reduction in energy intake by the proteinase inhibitor. Based on the results, the authors concluded that endogenous CCK may be have an important role in the control of food intake and that proteinase inhibition may have a potential for reducing food intake (Hill et al., 1990). Clinical trials on potato extracts containing Kunitz inhibitors showed no effect on satiety.
The efficiency of oral trypsin/chemotrypsin inhibitor in delaying the rate of gastric emptying in recently diagnosed type II diabetic patients and improving their post-prandial metabolic parameters have been examined (Schwartz, J. G., Guan, D., Green, G. M., Phillips, W. T. Treatment with an oral proteinase inhibitor slows gastric emptying and actually reduces glucose and insulin levels after a liquid meal in type II diabetic patients. Diabetes Care, 17: 255-262, 1994). Serum insulin, plasma glucose, plasma gastric inhibitory polypeptide levels, and the rate of gastric emptying were all significantly decreased over the 2 hour testing period in subjects who received proteinase inhibitor in their oral glucose/protein meal. U.S. Pat. No. 5,187,154 suggests the administration of CCK through an intramuscular injection or an intranasal spray. Alternatively, an oral administration of an agent that enhances endogenous release of CCK could represent an important approach to the treatment of Type 2 diabetes. One of the agents that may have a therapeutic application in patients with recently diagnosed Type 2 diabetes can be the potato proteinase inhibitor II.
Others have attempted to remove impurity proteins by the use of chromatography, including ion exchange, gel-filtration and affinity (Mellville et al.), ethanol protein solution (Bryant et al.), and precipitation with salt and solvents followed by dialysis (Pearce, G. and Ryan, C. A. A rapid, large-scale method for the purification of metallo-carboxypeptidase from potato tubers. Anal. Biochem. 30: 223-225, 1983). These methods can not be practiced feasibly on a production scale.
The invention consists of a process which utilizes heat treatment of potato proteins in the presence of salt, followed by centrifugation and filtration, as an efficient method for the elimination of Kunitz family, Bowman-Birk proteinase and carboxypeptidase inhibitors from other potato proteinase inhibitors. Raw potatoes are mixed with an organic acid, preferably formic acid, and a salt, preferably sodium chloride. The mixture is comminuted to reduce the size and increase the surface area of potato particles. The soluble proteins, including PI1, PI2, Kunitz family, Bowman-Birk and carboxypeptidase inhibitors, are released into the liquid phase and the mixture is centrifuged to remove solids.
The supernatant is incubated at a temperature of between about 60xc2x0 C. and about 80xc2x0 C., and preferably between about 70xc2x0 C. and 73xc2x0 C., for between about 30 minutes and about 180 minutes, and preferably between about 45 minutes and 75 minutes, to denature the impurity proteins without denaturing PI2. The solubility of the impurities was further reduced by lowering the temperature of the heat-treated material to between about 20xc2x0 C. and about 30xc2x0 C., and preferably between about 25xc2x0 C. and about 26xc2x0 C., at which temperatures PI2 remains soluble in the supernatant.
Centrifugation for 500,000 g-seconds or longer is used to remove the denatured impurity proteins from the heat-treated supernatant. Ultrafiltration using a cellulosic or sepharose membrane combined with diafiltration against an ammonium bicarbonate buffer is used to remove the carboxypeptidase inhibitor.
The process of the present invention is highly efficient in the separation and removal of Kunitz type inhibitor, previously found to interfere with the satiety efficacy of the PI2 in humans, as well as Bowman-Birk and carboxypeptidase inhibitors. The process also provides high recovery and yield of the PI2 inhibitor, increasing the concentration of PI2 in the final product by more than 100 times in comparison to its concentration in the proteins fraction in the raw potatoes. The process is efficient at laboratory, pilot plant and production scales, is easy to perform and does not require specialized equipment.